LOCUS       sequence-136117-        7187 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains the CORE cassette with convergent KlURA3 and hyg markers 
            and the I-SceI gene under the inducible GAL1 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7187)
  AUTHORS   Storici F, Durham CL, Gordenin DA, Resnick MA
  TITLE     Chromosomal site-specific double-strand breaks are efficiently 
            targeted for repair by oligonucleotides in yeast.
  JOURNAL   Proc Natl Acad Sci U S A. 2003 Dec 9;100(25):14994-9. Epub 2003 Nov 
            20.
  PUBMED    14630945
REFERENCE   2  (bases 1 to 7187)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl 
            Acad Sci U S A."; date: "2003-12-9"; volume: "100(25)"; pages: 
            "14994-9. Epub 2003 Nov 20"
COMMENT     Sequence Label: pGSHU
FEATURES             Location/Qualifiers
     source          1..7187
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(138..143,144..161,162..168)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    176..192
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     181..203
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     200..216
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     200..216
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        237..255
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     gene            complement(1843..3418)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     terminator      complement(1843..2040)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(2046..3074)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             complement(2046..3074)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     promoter        complement(3075..3418)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     promoter        complement(4200..4641)
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     primer_bind     complement(4206..4229)
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     protein_bind    4524..4641
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     complement(4930..4947)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5101..5689)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5181..5200)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5860..6651,6652..6720))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6483..6502
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6721..6825)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6893..6911
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6949..6971)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7071..7090
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(7171..7187,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(7171..7187,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg acggatcccc ctggcacgac
       61 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttacctcact
      121 cattaggcac cccaggcttt acactttatg cttccggctc ctatgttgtg tggaattgtg
      181 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gctcggaatt
      241 aaccctcact aaagggaaca aaagctggta ccgggcccgg agacaatcat atgggagaag
      301 caattggaag atagaaaaaa ggtactcggt acataaatat atgtgattct gggtagaaga
      361 tcggtctgca ttggatggtg gtaacgcatt tttttacaca cattacttgc ctcgagcatc
      421 aaatggtggt tattcgtgga tctatatcac gtgatttgct taagaattgt cgttcatggt
      481 gacactttta gctttgacat gattaagctc atctcaattg atgttatcta aagtcatttc
      541 aactatctaa gatgtggttg tgattgggcc attttgtgaa agccagtacg ccagcgtcaa
      601 tacactcccg tcaattagtt gcaccatgtc cacaaaatca tataccagta gagctgagac
      661 tcatgcaagt ccggttgcat cgaaactttt acgtttaatg gatgaaaaga agaccaattt
      721 gtgtgcttct cttgacgttc gttcgactga tgagctattg aaactagttg aaacgttggg
      781 tccatacatt tgccttttga aaacacacgt tgatatcttg gatgatttca gttatgaggg
      841 tactgtcgtt ccattgaaag cattggcaga gaaatacaag ttcttgatat ttgaggacag
      901 aaaattcgcc gatatcggta acacagtcaa attacaatat acatcgggcg tttaccgtat
      961 cgcagaatgg tctgatatca ccaacgccca cggggttact ggtgctggta ttgttgctgg
     1021 cttgaaacaa ggtgcgcaag aggtcaccaa agaaccaagg ggattattga tgcttgctga
     1081 attgtcttcc aagggttctc tagcacacgg tgaatatact aagggtaccg ttgatattgc
     1141 aaagagtgat aaagatttcg ttattgggtt cattgctcag aacgatatgg gaggaagaga
     1201 agaagggttt gattggctaa tcatgacccc aggtgtaggt ttagacgaca aaggcgatgc
     1261 attgggtcag cagtacagaa ccgtcgacga agttgtaagt ggtggatcag atatcatcat
     1321 tgttggcaga ggacttttcg ccaagggtag agatcctaag gttgaaggtg aaagatacag
     1381 aaatgctgga tgggaagcgt accaaaagag aatcagcgct ccccattaat tatacaggaa
     1441 acttaataga acaaatcaca tatttaatct aatagccacc tgcattggca cggtgcaaca
     1501 ctcacttcaa cttcatctta caaaagatca cgtgatctgt tgtattgaac tgaaaatttt
     1561 ttgtttgctt ctctctctct ctttcattat gtgagagttt aaaaaccaga aactacatca
     1621 tcgaaaaaga gtttaaacca ttacaaccat tgcgataagc cctctcaaac ttcctccaga
     1681 ctgtttgtca tccaattggt aagattaatt atcatatacc ccggataaga aaagaagcaa
     1741 gcacacaatg gatcaactta atggtaagga acaacaagcg gccgccaccg cggtggagct
     1801 cgttttcgac actggatggc ggcgttagta tcgaatcgac agcagtatag cgaccagcat
     1861 tcacatacga ttgacgcatg atattacttt ctgcgcactt aacttcgcat ctgggcagat
     1921 gatgtcgagg cgaaaaaaaa tataaatcac gctaacattt gattaaaata gaacaactac
     1981 aatataaaaa aactatacaa atgacaagtt cttgaaaaca agaatctttt tattgtcagt
     2041 actgattatt cctttgccct cggacgagtg ctggggcgtc ggtttccact atcggcgagt
     2101 acttctacac agccatcggt ccagacggcc gcgcttctgc gggcgatttg tgtacgcccg
     2161 acagtcccgg ctccggatcg gacgattgcg tcgcatcgac cctgcgccca agctgcatca
     2221 tcgaaattgc cgtcaaccaa gctctgatag agttggtcaa gaccaatgcg gagcatatac
     2281 gcccggagcc gcggcgatcc tgcaagctcc ggatgcctcc gctcgaagta gcgcgtctgc
     2341 tgctccatac aagccaacca cggcctccag aagaagatgt tggcgacctc gtattgggaa
     2401 tccccgaaca tcgcctcgct ccagtcaatg accgctgtta tgcggccatt gtccgtcagg
     2461 acattgttgg agccgaaatc cgcgtgcacg aggtgccgga cttcggggca gtcctcggcc
     2521 caaagcatca gctcatcgag agcctgcgcg acggacgcac tgacggtgtc gtccatcaca
     2581 gtttgccagt gatacacatg gggatcagca atcgcgcata tgaaatcacg ccatgtagtg
     2641 tattgaccga ttccttgcgg tccgaatggg ccgaacccgc tcgtctggct aagatcggcc
     2701 gcagcgatcg catccatggc ctccgcgacc ggctgcagaa cagcgggcag ttcggtttca
     2761 ggcaggtctt gcaacgtgac accctgtgca cggcgggaga tgcaataggt caggctctcg
     2821 ctgaattccc caatgtcaag cacttccgga atcgggagcg cggccgatgc aaagtgccga
     2881 taaacataac gatctttgta gaaaccatcg gcgcagctat ttacccgcag gacatatcca
     2941 cgccctccta catcgaagct gaaagcacga gattcttcgc cctccgagag ctgcatcagg
     3001 tcggagacgc tgtcgaactt ttcgatcaga aacttctcga cagacgtcgc ggtgagttca
     3061 ggctttttac ccatggttgt ttatgttcgg atgtgatgtg agaactgtat cctagcaaga
     3121 ttttaaaagg aagtatatga aagaagaacc tcagtggcaa atcctaacct tttatatttc
     3181 tctacagggg cgcggcgtgg ggacaattca acgcgtctgt gaggggagcg tttccctgct
     3241 cgcaggtccg cagcgaggag ccgtaatttt tgcttcgcgc cgtgcggcca tcaaaatgta
     3301 tggatgcaaa tgattataca tggggatgta tgggctaaat gtacgggcga cagtcacatc
     3361 atgcccctga gctgcgcacg tcaagactgt caaggagggt attctgggcc tccatgtcgc
     3421 tggccgggtg acccggcggg gacgaggcaa gctaaacaga tctgacttat tatttcagga
     3481 aagtttcgga ggagatagtg ttcggcagtt tgtacatcat ctgcgggatc aggtacggtt
     3541 tgatcaggtt gtagaagatc aggtaagaca tagaatcgat gtagatgatc ggtttgtttt
     3601 tgttgatttt tacgtaacag ttcagttgga atttgttacg cagaccctta accaggtatt
     3661 ctacttcttc gaaagtgaaa gactgggtgt tcagtacgat cgatttgttg gtagagtttt
     3721 tgttgtaatc ccatttacca ccatcatcca tgaaccagta tgccagagac atcggggtca
     3781 ggtagttttc aaccaggttg ttcgggatgg tttttttgtt gttaacgatg aacaggttag
     3841 ccagtttgtt gaaagcttgg tgtttgaaag tctgggcgcc ccaggtgatt accaggttac
     3901 ccaggtggtt aacacgttct tttttgtgcg gcggggacag tacccactga tcgtacagca
     3961 gacatacgtg gtccatgtat gctttgtttt tccactcgaa ctgcatacag taggttttac
     4021 cttcatcacg agaacggatg taagcatcac ccaggatcag accgatacct gcttcgaact
     4081 gttcgatgtt cagttcgatc agctgggatt tgtattcttt cagcagttta gagttcggac
     4141 ccaggttcat tacctggttt tttttgatgt ttttcatatg catggatccg gggttttttc
     4201 tccttgacgt taaagtatag aggtatatta acaatttttt gttgatactt ttattacatt
     4261 tgaataagaa gtaatacaaa ccgaaaatgt tgaaagtatt agttaaagtg gttatgcagt
     4321 ttttgcattt atatatctgt taatagatca aaaatcatcg cttcgctgat taattacccc
     4381 agaaataagg ctaaaaaact aatcgcatta tcatcctatg gttgttaatt tgattcgttc
     4441 atttgaaggt ttgtggggcc aggttactgc caatttttcc tcttcataac cataaaagct
     4501 agtattgtag aatctttatt gttcggagca gtgcggcgcg aggcacatct gcgtttcagg
     4561 aacgcgaccg gtgaagacga ggacgcacgg aggagagtct tccttcggag ggctgtcacc
     4621 cgctcggcgg cttctaatcc gtacttcaat atagcaatga gcagttaagc gtattactga
     4681 aagttccaaa gagaaggttt ttttaggcta agataatggg gctctttaca tttccacaac
     4741 atataagtaa gattagatat ggatatgtat atggatatgt atatggtggt aatgccatgt
     4801 aatatgatta ttaaacttct ttgcgtccat ccaacgagat ctggcgcgcc ttaattaacc
     4861 caacctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     4921 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     4981 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     5041 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     5101 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     5161 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     5221 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     5281 cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     5341 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     5401 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     5461 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     5521 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
     5581 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     5641 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     5701 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     5761 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     5821 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     5881 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     5941 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     6001 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     6061 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     6121 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     6181 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     6241 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     6301 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     6361 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     6421 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     6481 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     6541 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     6601 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     6661 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     6721 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     6781 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     6841 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     6901 tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     6961 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     7021 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
     7081 gcagattgta ctgagagtgc accatatgga catattgtcg ttagaacgcg gctacaatta
     7141 atacataacc ttatgtatca tacacatacg atttaggtga cactata
//
